Luciferases catalyze light emission in the presence of their substrates, luciferins, and this property has made them a staple in multiple basic scientific research applications ranging from quantitative analysis of promoter activity and cell viability and proliferation in cultured cells to non-invasive bioluminescence imaging of biological processes such as cell tracking, tumor growth kinetics and response to therapy in vivo.1-3 There are different types of luciferases that occur in species including beetles, bacteria, worms, fungi, and squid with several of them cloned and tested for molecular biology research.4, 5 Each of these luciferases has different characteristics which makes them attractive for certain applications but not optimal for others. For high-throughput applications, an optimal luciferase would display the following characteristics: (1) enzyme stability over a variety of conditions; (2) high light output for increased sensitivity; (3) non-invasive monitoring of enzymatic activity at different time points in real-time; and (4) the catalysis of stable light emission for minimal variability between thousands of screened wells.
The marine copepod, Gaussia princeps, secretes a luciferase (Gluc) which possesses all but the fourth characteristic. In recent years, the cloned cDNA of Gluc has been shown to be the preferred luciferase type for many different biological applications.6-13 Gluc is the smallest luciferase cloned (18 kDa) with several advantages over other commonly used reporters: Gluc is over 2,000-fold more sensitive than firefly (Fluc) or Renilla (Rluc) luciferases and 20,000-fold more sensitive than the secreted alkaline phosphatase;6,14 Gluc is naturally secreted and therefore monitoring of biological processes can be accomplished in real-time by measuring enzymatic activity in aliquots of conditioned medium in cultured cells at different time points keeping the cells intact for confirmation analysis making it useful for studying assay kinetics;6,15 it is stable over a wide pH range and in the presence of reactive compounds;14,16 in vivo, Gluc can be detected in blood or urine making it a sensitive ex-vivo tool for monitoring of in vivo processes.13 One limitation of Gluc for high-throughput assays is the rapid decay of its bioluminescence reaction and therefore a luminometer with a built-in injector is required, making the assay time consuming.14 